Quantitative optical microscopy for aberrating and scattering biological media

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Optical microscopy in biology develops today along two major directions: improving imaging quality, especially in terms of resolution and 3D field of view; and improving content and quantitativeness of associated measurements, such as molecular concentration and dynamics. For obtaining the latter information, a very successful and well-established technique is Fluorescence Fluctuation Microscopy (FFM), which is a generalisation of classical Fluorescence Correlation Spectroscopy (FCS) that exploits different microscopy techniques. Within this context, the main goals of the MICROSCATTAB project are: i) to understand how light scattering and optical aberrations disturb measurements performed with Fluorescence Fluctuation Microscopy; ii) to develop new schemes to correct for these effects. In the past, Adaptive Optics (AO) approaches have been successfully used in microscopy for correcting sample-induced aberrations, but the majority of these methods have been concerned with compensating low-order aberrations which arise in rather transparent samples or at shallow focusing depth. However, when focusing deep into a specimen (tens to hundreds of µm), large-amplitude aberrations with complex phase structure arise, and light scattering dramatically increases. In our project, we will investigate how to take these effects into account, and what are the most efficient correction schemes for FFM. For achieving this, we will perform experimental measurements on aberrating/scattering phantoms combined with analytical and numerical modelling of the experiments. The resulting wavefront correction schemes will then be applied to FFM in a biological tissue model.